This invention relates to nucleic acid and amino acid sequences of a novel human anion channel and to the use of these sequences in the diagnosis, prevention, and treatment of cancer and developmental disorders.
Chloride channels are found in the plasma membranes of virtually every cell in the body. Chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH (cf. Greger, R. (1988) Annu. Rev. Physiol. 50:111-122). Electrophysiological and pharmacological measurements including ion conductance, current-voltage relationships, and sensitivity to modulators suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
Several chloride channels have been cloned from mammalian tissues and cell lines. The sequences of these proteins are diverse and include a nicotinic acetylcholine receptor homolog, the cystic fibrosis transmembrane conductance regulator, and the highly acidic p64 protein with no significant homology to other proteins (Bernard, E. A. et al. (1987) Trends Neurosci. 16:502-509; Riordan, J. R. et al. (1989) Science 245:1066-1073; Landry, D. et al. (1993) J. Biol. Chem. 268:14948-14955). Many of the channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II which regulate chloride channel activity in epithelial cells.
The p64 protein was originally identified in bovine kidney cortex membranes by its affinity for indanyloxyacetic acid, a known inhibitor of epithelial chloride channels (Landry et al. (1989) Science 244:1469-1472). Antibodies raised against the isolated p64 protein can deplete solubilized kidney membranes of all detectable chloride channel activity. Thus, p64 is likely to be a functional component of the kidney chloride channel (Redhead, R. C. et al. (1992) Proc. Natl. Acad. Sci. 89:3716-3720).
Northern blot analyses using the p64 clone as a probe detect related mRNAs, ranging in size from xcx9c2 kb to xcx9c6.5 kb, in bovine kidney cortex, kidney medulla, liver, adrenal glands, brain, skeletal muscle, and heart. Most of these tissues have multiple transcripts that are capable of hybridizing to this probe. The diversity and relative abundance of these transcripts is tissue-specific, and this suggests that the p64 transcripts are alternatively spliced and/or that a family of related genes are expressed (Landry et al., supra).
The sequence of p64 predicts an acidic, integral membrane protein which spans the membrane at least twice and has the amino terminus on the cytoplasmic side. The protein has potential sites for phosphorylation by protein kinase C, tyrosine kinase, and casein kinase II, and a single site for N-linked glycosylation at Asp235.
The discovery of polynucleotides encoding a novel human anion channel, and the molecules themselves, provides a means to investigate the regulation of membrane potentials, intracellular pH, cell volume, signal transduction, and transepithelial ion transport in tissues containing absorptive or secretory epithelia under normal and disease conditions. Discovery of a novel anion channel satisfies a need in the art by providing new compositions useful in diagnosing and treating cancer and developmental disorders.
The present invention features a novel human anion channel protein hereinafter designated NANCH and characterized as having similarity to bovine p64 chloride channel and human P64CLCP.
Accordingly, the invention features a substantially purified NANCH having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode NANCH. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode NANCH. The present invention also features antibodies which bind specifically to NANCH, and pharmaceutical compositions comprising substantially purified NANCH. The invention also features the use of agonists and antagonists of NANCH. The invention also features methods for treating disorders which are associated with NANCH, and for detecting a polynucleotide which encodes NANCH.